SKU: WT229E Category:

U-Blot® Oxidated Insulin ELISA Kit

$999.00

SKU: WT229E
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Product details

The Oxidated Insulin ELISA Kit uses the “biotin probes” method to biotin label carbonyl groups of oxidized proteins. In this method, the biotin probes first react with carbonyl groups of oxidized proteins to form unstable Schiff bases, which are then further reduced to more stable amines. The biotinylated proteins can then be detected by ELISA. An anti-pan Insulin antibody has been coated onto a 96-well plate. Biotinylated samples are pipetted into the wells, and Insulin present in a sample is bound to the wells by the immobilized antibody. The wells are washed, and HRP-conjugated streptavidin is used to detect oxidated Insulin. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of oxidated Insulin bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.This assay enables quick and easy quantification of target proteins and allows high-throughput screening of numerous different lysates without the need for traditional Western Blot analysis. It features minimal hands-on operation time, simple workflow, and completely non-radioactive detection system, ensuring safe and convenient experimental operation.

Specifications

Size1 Plate Kit, 2 Plate Kit, 5 Plate Kit
SpeciesHuman
Compatible Sample TypesTissue Lysates, Cell Lysates
Accession NumberP01308
Gene Id3630
Gene SymbolsINS
Protein Name / SynonymsInsulin
Solid Support96-well Microplate
Design PrincipleSandwich-based
Method Of DetectionColorimetric
Research AreaPost-Translational Modifications, Epigenetics
ModificationOxidation
Shipping Type2‑8 ℃
Storage-20°C
Kit ComponentsMicroplate Coated With Anti-Pan-Insulin Antibody:1 plate;Positive Control:1 vial;HRP-Conjugated Streptavidin:200 μL;Wash Buffer:25 mL;Assay Diluent:15 mL;Lysis Buffer:5 mL;TMB One-Step Substrate Reagent:12 mL;Stop Solution:8 mL;Oxidation Buffer A:12 mL;Oxidation Buffer B:3 mL;Oxidation Labeling Reagent:1 vials;Oxidation Stabilizing Reagent:1 vials;
Additional Materials Required20% (v/v) trichloroacetic acid (TCA) solution, ice-cold;1:1 (v/v) ethanol:ethyl acetate;Benchtop centrifuge and microcentrifuge (4°C);Microplate reader capable of measuring absorbance at 450 nm;Protease and Phosphatase inhibitors;Precision pipettes to deliver 2 μL to 1 mL volumes;Adjustable 1-25 mL pipettes for reagent preparation;100 mL and 1 liter graduated cylinders;Log-log graph paper or computer and software for ELISA data analysis;Absorbent paper;Tubes to prepare positive control or sample dilutions;Distilled or deionized water;Shaker
Assay Procedure SummaryPrepare all reagents, samples and Positive Control as instructed; Label samples with Oxidation Labeling Reagent;Add Oxidation Stabilizing Reagent to samples; Precipitate proteins with ice-cold 20% TCA; Wash protein pellet twice with 1:1 (v/v) ethanol:ethyl acetate; Resuspend protein pellets in Oxidation Buffer B; Add 100 μL sample and Positive Control to each well. Incubate 2.5 hours at room temperature or overnight at 4°C with gentle shaking; Add 100 μL prepared HRP-Conjugated Streptavidin solution. Incubate for 1 hour at room temperature with gentle shaking; Add 100 μL TMB One-Step Substrate Reagent to each well. Incubate 30 minutes at room temperature; Add 50 μL Stop Solution to each well. Read at 450 nm immediately.
Storage/StabilityThe entire kit may be stored at -20°C for up to 6 months from the date of shipment. Avoid repeated freeze-thaw cycles.
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